Method for the characterization of nucleic acid molecules involving generation of extendible upstream DNA fragments resulting from the cleavage of nucleic acid at an abasic site

ABSTRACT

The present invention is drawn to a method for characterising nucleic acid molecules, which comprises the steps of: i) introducing a modified base which is a substrate for a DNA glycosylase into a DNA molecule; ii) excising the modified base with the DNA glycosylase to generate an abasic site; iii) cleaving the DNA at the abasic site to generate and release an extendible upstream DNA fragment having a 3′ hydroxyl terminus; and iv) incubating the released extendible upstream DNA fragment in the presence of an enzyme allowing for extension thereof and an additional template nucleic acid and analysing resultant fragment(s).

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/IE98/00030 which has an Internationalfiling date of Apr. 22, 1998, which designated the United States ofAmerica.

TECHNICAL FIELD

The present invention relates to a method for characterising nucleicacid molecules which involves generating extendible upstream DNAfragments which result from the cleavage of nucleic acid at an abasicsite.

BACKGROUND ART

Characterisation of target nucleic acids is highly important for severalreasons relating to confirmation of the presence or absence of a gene ina sample, confirmation of part or all of a nucleic acid sequence, andscreening for the presence of known and unknown disease causingmutations leading to inherited disease and natural variations in DNA.Although there are many known methods for characterising nucleic acidand for the detection of unknown sequence changes, the increasing amountof new genetic information being generated makes it important to developnew, better and faster methods for characterisation of nucleic acids.

WO 97/03210 discloses the use of a DNA-glycosylase enzyme, whichrecognises a modified base, for the direct detection of known andunknown mutations in a target nucleic acid sample. The method typicallyinvolves amplifying a target nucleic acid sample using a combination ofnormal DNA precursor nucleotides and one or more modified precursornucleotide(s) where the modified precursor nucleotide replaces one ofthe normal precursor nucleotides which is a substrate for a DNAglycosylase. Following excision of the modified base by the glycosylase,the resulting abasic site is cleaved and the products of the cleavageare analysed. This method allows detection of mutations at candidateloci. However, the method of WO 97/03210 has certain limitations. Forexample, with this method it is not possible to detect sequencedifferences between nucleic acid molecules without detecting sequencesimilarities and thus multiple samples cannot be combined forsimultaneous analysis.

DISCLOSURE OF INVENTION

The invention provides a method for characterising nucleic acidmolecules, which comprises the steps of:

i) introducing a modified base which is a substrate for a DNAglycosylase into a DNA molecule;

ii) excising the modified base by means of said DNA glycosylase so as togenerate an abasic site;

iii) cleaving the DNA at the abasic site so as to generate an upstreamDNA fragment that can be extended; and

iv) incubating the extendible upstream fragment in the presence of anenzyme allowing for extension thereof and a template nucleic acid andanalysing the resultant fragment(s).

The invention provides a novel, versatile and simple method using theabove-mentioned extendible upstream DNA fragments which allowscharacterisation of nucleic acids and which has advantages over existingmethods as indicated in the following description.

One of the most important uses (but not the only use) of the methodaccording to the invention is to scan or check a fragment of DNA (targetnucleic acid) for the presence or absence of a mutation. The methodessentially consists of i) the generation of the extendible upstream DNAfragments and ii) the subsequent use of these fragments in analysing apiece of DNA (e.g. detecting a mutation).

Preferably, the modified base used is uracil, hypoxanthine or 8-OHguanine.

Preferably, the modified bases are derived from modified precursornucleotides which when incorporated into DNA generate said modifiedbases.

Thus, the preferred modified precursor nucleotides are dUTP, dITP and8-OH dGTP which when incorporated into DNA generate the glycosylasesubstrate bases uracil, hypoxanthine and 8-OH guanine, respectively.Each of the modified precursor nucleotides is a base sugar phosphatecomprising said base and a sugar phosphate moiety. Uracil in DNA isrecognised specifically by uracil DNA-glycosylase (UDG) and releasedfrom DNA. UDG also recognises other uracil related bases when present inDNA. Hypoxanthine is recognised specifically by alkylpurineDNA-glycosylases (ADG) and released from DNA. This enzyme alsorecognises and releases N3 methyladenine, N3 methylguanine, O²methylcytosine and O² methylthymine when present in DNA. 8-OH guanine isrecognised specifically by formamido-pyrimidine DNA-glycosylase (FPG)and released from DNA. This enzyme also recognises and releases ringopened purines when present in DNA. Thymine DNA glycosylase recognisesand releases uracil and thymine positioned opposite guanine bases inDNA.

Modified precursor nucleotide(s) as used herein refers to a modifiednucleotide or nucleotides that can be incorporated into a nucleic acidso that a modified base or bases is generated which is/are recognisedand can be excised by a DNA glycosylase enzyme.

Following the introduction of the modified base, the DNA product istreated with a suitable DNA glycosylase enzyme which recognises andreleases the glycosylase substrate base present in the target sample andconsequently generates an apurinic or apyrimidinic (AP) site, dependingon the nature of the modified base(s). AP site is the term given to asite in DNA where the base moiety of a nucleotide has been lost orremoved, leaving behind a deoxyribophosphate with the DNA phosphodiesterbackbone still intact. AP is the abbreviation for either apurinic and/orapyrirmidinic depending on whether a purine or pyrimidine base had beenattached to the ribose ring. An AP site is also referred to as an abasicsite, being the general term for apurinic and apyrimidinic site.

Release of the glycosylase substrate base from the nucleic acid sampleresults for example in an apyrimidinic site in the case of uracil and anapurinic site in the case of hypoxanthine and 8-OH dG. Collectively,such sites are referred to as abasic sites.

Essentially, cleaved fragments must have hydroxyl groups at the 3′termini and the DNA immediately downstream of said 3′ termini must notbe blocked in a way that prevents extension of the fragment from said 3′termini. Cleaved fragments are generated that have hydroxyl at the 3′termini and downstream blocking groups are removed that preventextension of the fragment from the said 3′ termini, while using astemplate, that DNA from which the extendible fragment was derived.

The DNA can be cleaved in a number of ways at the abasic site so as togenerate said upstream DNA fragment as hereinafter described in greaterdetail.

For example, the phosphate linkages at the abasic sites can be cleavedby a treatment selected from treatment with a basic solution or otherchemical treatment, heat treatment and/or treatment with an enzyme.

According to one embodiment of the invention, the upstream fragment isgenerated by cleaving the DNA at the 5′ side of the abasic site suchthat the 3′ terminus of the upstream fragment bears a hydroxyl group.

The terms extendible fragment and upstream DNA fragment are usedinterchangeably herein.

As the 3′-OH termini are thereby generated no further processing of theupstream fragments is required prior to step iv).

Treatment with basic solutions (alkali) at high temperature or with achemical such as piperidine, or with an enzyme which cuts specificallyat abasic sites, such as E. coli endonuclease IV results in cleavage ofthe abasic site on the 5′ side.

Suitably in this embodiment, the cleavage is achieved with a 5′ APendonuclease.

According to an alternative embodiment the upstream fragment isgenerated by cleaving at the 5′ side of the abasic site so as to leave aphosphate group at the 3′ terminus of the upstream fragment and removingthe phosphate group so that the upstream fragment bears a hydroxyl groupat the 3′ terminus.

Alternatively, cleavage with alkali or the abasic endonuclease activityof FPG is used, followed by removal of the 3′ phosphate. For example 3′phosphate groups can be removed by enzymatic means using enzymes with 3′phosphate activity such as T4 polynucleotide kinase.

According to a still further embodiment, the upstream fragment isgenerated by cleaving at the 3′ side of the abasic site so as togenerate a deoxyribose group phosphate at the 3′ terminus of theupstream fragment and subsequently removing the deoxyribose group toleave a hydroxyl group at the 3′ terminus.

This can be achieved by using an enzyme with 3′deoxyribo-phosphodiesterase activity, or using FPG followed by a 3′phosphatase.

In an alternative embodiment 5′ deoxyribose moieties downstream of the3′ terminus of the upstream fragment are removed so that the upstreamfragment can be extended on the template.

Preferably the 5′ deoxyribose moieties are removed by a 5′deoxyribophosphodiesterase.

Treatment with high temperature alone or a 3′ AP endonuclease results incleavage of the abasic site to completion on the 3′ side.

Glycosylase mediated cleavage cuts the extended primer at an abasic sitesubsequent to release of the modified base by a DNA glycosylase yielding3′ termini with 3′-OH or 3′ phosphate groups or deoxyribophosphategroups. Except in cases where 3′-OH termini are generated, all othertermini require further processing prior to extension of the upstreamfragment.

Glycosylase mediated cleavage in the method according to the inventionrefers to both 5′ and 3′ cleavage, including whatever subsequenttreatment that is necessary to generate a 3′ OH group at the 3′ terminusof the upstream fragment.

Preferably, the modified base is introduced by enzymatic amplificationof the DNA.

Preferably, the DNA (herein also referred to as target nucleic acid) isamplified using normal DNA precursor nucleotides and at least onemodified precursor nucleotide.

Precursor nucleotides in the case of a DNA amplification process referto the deoxyribonucleotides dATP, dCTP, dGTP and dTTP herein referred toas “normal” DNA precursor nucleotides.

The term “amplifying” as used herein refers to any in vitro process forincreasing the number of copies of a nucleotide sequence or sequences.Amplification of a target nucleic acid molecule results in theincorporation of precursor nucleotides into the DNA being amplified.Typically, amplification of a target sample is carried out usingappropriate primers in the polymerase chain reaction (PCR). During theamplification, primers anneal to the target nucleic acid and areextended using a DNA polymerase in a 5′ to 3′ direction on the targetnucleic acid which acts as a template for synthesis of new DNA. The useof flanking primers, which are referred to herein as initial primers,which anneal to the upper and lower-strands of a DNA molecule permitexponential amplification of the DNA segment delimited by the upper andlower primers.

The amplification will typically involve amplifying a target nucleicacid using a combination of normal DNA precursor nucleotides and one ormore modified precursor nucleotide(s) where the modified precursornucleotide replaces all or a proportion of one of the normal precursornucleotides. Amplification of a nucleic acid using normal DNA precursornucleotides results in the incorporation of the four normal bases G,A,T,or C into DNA. Amplification of a nucleic acid using a modifiedprecursor nucleotide in place of one of the normal precursor nucleotidesresults in the incorporation of a glycosylase substrate base into DNA inplace of one of the four normal bases G,A,T, or C.

The target nucleic acid sample will typically be DNA. However, RNA mayalso be used following conversion to DNA by reverse transcription.

When a modified precursor nucleotide replaces a proportion of one of thenormal precursor nucleotides, the ratio of the modified precursornucleotide to the normal precursor nucleotide that it is replacing issuch that an optimum of one modified precursor nucleotide isincorporated per strand of amplified DNA. This allows subsequentcleavage of the amplified DNA strand into two fragments followingcontact with a DNA glycosylase and an abasic site cleavage agent asherein described. This method of cleavage is herein referred to asglycosylase mediated cleavage. A higher ratio of the modified precursornucleotide to normal precursor nucleotide is used to generate more thanone cleavage site per amplified DNA strand. The incorporation of amodified precursor nucleotide into the amplified product generates oneor more modified bases at one or more positions recognised by a DNAglycosylase enzyme in the amplified product.

Replacement of all of a normal precursor nucleotide with a modifiedprecursor nucleotide in the amplification reaction if used in step i)permits glycosylase mediated cleavage of a primer extended in anamplification reaction at the first position 3′ of the extended primerwhere a normal base is replaced by a modified base. Thus, if thetemplate sequence immediately 3′ of a location where the primerhybridises is CTAG and the modified nucleotide precursor is dUTPreplacing dTTP, then the modified base uracil (U) will be incorporatedopposite A on the template strand. Thus, in this situation, the primerwill have been extended by two nucleotides at the 3′ end (primer-GA 3′)following amplification and glycosylase mediated cleavage. Theseextended primers generated following the initial extension andglycosylase mediated cleavage are referred to herein inter alia asextendible fragments, as indicated above, whereas the primers prior toextension are referred to as the initial primers herein.

The extended 3′ terminal sequence of the extendible fragment isenzymatically synthesised and is directly related to the nucleic acidbeing characterised as the nucleic acid acts as the template for itssynthesis. Thus the 3′ end of the extendible fragment is complementaryto the nucleic acid. Accordingly, determination of the nature of the 3′end of the extendible fragment by any means allows characterisation ofthe nucleic acid from which it was derived. If an initial primer isplaced adjacent to a locus where a DNA sequence variation such as apolymorphism or a mutation occurs so that the first modified baseincorporated into the extended primer is at the mutation locus, then theinitial primer will be extended to a different length depending onwhether or not a mutation is present at the mutation locus followingglycosylase mediated cleavage. The extendible fragments are subsequentlytreated (if necessary) so that they can be used as primers for asubsequent extension reaction. Because the sequence at the 3′ termini ofthe extendible fragments differ depending on whether a mutation ispresent or absent at the mutation locus, analysis of the ability of anextendible fragment to function in a subsequent extension reaction usinga template nucleic acid permits the determination of whether a mutationis present or absent at the mutation locus. Any naturally occurring orenzymatically or chemically synthesised template which fully orpartially hybridises to the extendible fragment can be designed and/orselected as a template nucleic acid allowing the ability of theextendible fragment to function as a primer to be determined.

When a proportion of a normal precursor nucleotide is replaced with amodified precursor nucleotide in the amplification reaction, glycosylasemediated cleavage of the primer extended in an amplification reactionwill yield a population of extendible fragments of various lengths sincedifferent molecules will be cleaved at different points depending onwhere the modified precursor nucleotide is incorporated. The length ofeach fragment is determined by the position of incorporation of themodified precursor nucleotide during extension from the 3′ end of theinitial primer.

Amplification of a target nucleic acid using the precursor nucleotidesdATP, dCTP, dGTP, dTTP and a low amount of the modified precursornucleotide dUTP results in an amplified DNA where thymine is replacedrandomly by uracil. The uracil is incorporated in the newly synthesisedDNA strand at positions complementary to adenine residues in thetemplate DNA strand during the amplification process. Amplification of atarget nucleic acid using the precursor nucleotides dATP, dCTP, dGTP,dTTP and a low amount of the modified precursor nucleotide dITP resultsin an amplified DNA where guanine is preferentially replaced randomly byhypoxanthine. The hypoxanthine is incorporated in the newly synthesisedDNA strand at positions complementary to cytosine residues in thetemplate DNA strand during the amplification process when the otherprecursor nucleotides are not limiting. Amplification of a targetnucleic acid using the precursor nucleotides dATP, dCTP, dGTP, dTTP anda low amount of the modified precursor nucleotide 8-OH dGTP results inan amplified DNA where guanine is preferentially replaced randomly by8-OH guanine. The 8-OH guanine is incorporated in the newly synthesisedDNA strand at positions complementary to cytosine residues in thetemplate DNA strand during the amplification process when the otherprecursor nucleotides are not limiting.

The amplified DNA strands can be separated for separate analysis of therespective strands. In addition, the separated strands can beimmobilised, which can be achieved by several means. A common method forimmobilisation and/or separation of DNA strands is by the use of thebiotin streptavidin interaction, where normally, the DNA contains thebiotin label and the streptavidin is attached to a solid support.However, the method according to the invention in its various steps isamenable to immobilisation formats that allows immobilisation of theupstream fragment, the strand bearing the upstream fragment, the strandcomplementary to the strand bearing the upstream fragment, the templatenucleic acid, the target nucleic acid and the products generated fromglycosylase mediated cleavage of amplified or extended nucleic acidsbearing modified bases.

The modified base can be introduced by chemical modification of anucleic acid, rather than by an amplification technique such as PCR.

Several methods exist where the treatment of DNA with specific chemicalsmodifies existing bases so that they are recognised by specific DNAglycosylase enzymes. For example, treatment of DNA with alkylatingagents such as methylnitrosourea generates several alkylated basesincluding N3-methyladenine and N3-methylguanine that are recognised andexcised by alkyl purine DNA-glycosylase. Treatment of DNA with sodiumbisulfite causes deamination of cytosine residues in DNA to form uracilresidues which are recognised and excised by uracil DNA-glycosylase.Treatment of DNA with ferrous sulphate and EDTA causes oxidation ofguanine residues in DNA to form 8-OH guanine residues in DNA which arerecognised and excised by formamido-pyrimidine DNA glycosylase.

Thus, bases present in the nucleic acid or indeed, the extendibleupstream fragment generated in step iii) can be converted into modifiedbases by chemical means. A proportion or all of the cytosine residuescan be readily converted to an uracil using sodium bisulfite therebyrendering the amplified sample susceptible to uracil DNA-glycosylasecleavage at the sites of cytosine conversion. If the upper or lowerprimer is synthesised so that it contains 5-methylcytosine rather thancytosine, in such a case the primer will be resistant to uracilDNA-glycosylase mediated cleavage since deamination of 5-methylcytosineoccurs at a reduced rate by comparison with cytosine and generates athymine rather than an uracil residue.

Prior to treatment with a suitable DNA glycosylase, double stranded DNAmay be treated with exonuclease I. This treatment serves to digest theunused primers and any non-specific single stranded DNA amplificationproducts thus improving the signal to noise ratio.

In the case where the modified precursor nucleotide is dUTP, themodified base uracil will be generated at thymine positions in theamplified target nucleic acid sample. Addition of uracil DNA-glycosylaseto the sample releases the uracil from the sample. In the case where themodified precursor nucleotide is dITP, the modified base hypoxanthinewill be generated at guanine positions in the amplified target nucleicacid sample. Addition of alkylpurine DNA-glycosylase to the samplereleases the hypoxanthine from the sample. In the case where themodified precursor nucleotide is 8-OH dGTP, the modified base 8-OHguanine will be generated at guanine positions in the amplified targetnucleic acid sample. Addition of formamido-pyrimidine DNA-glycosylase tothe sample releases the 8-OH guanine from the sample.

Suitably a primer or one or more nucleotide(s) involved in the enzymaticamplification is labelled.

The initial primer used may be suitably labelled. Labelling of theprimers can be performed by a variety of means including addition of aradioactive, fluorescent, or detectable ligand to the primer during orpost primer synthesis.

In one embodiment of the invention, the enzyme used in step iv) is apolymerase which can be incubated with the extendible upstream fragmentin the presence of one or more nucleotide(s).

Also in this embodiment suitably one or more of the nucleotide(s) ofstep iv) is a dideoxy nucleotide.

Also one or more of the nucleotides of step iv) can be labelled.

Various nucleic acid polymerases can be used to extend the 3′ terminusof the extendible fragment on a template nucleic acid. Many polymerasesare described in the literature that extend 3′ termini of primers on atemplate DNA. For example, DNA polymerases isolated from phage andmesophilic and thermophilic bacteria can be used.

Several DNA polymerases, including T7 DNA polymerase incorporate dideoxyterminator nucleotides as a well as normal precursor nucleotides duringextension of primers. Thermophilic DNA polymerases are used routinely inamplification of nucleic acids through repeated cyclic extension ofprimers. The upstream DNA fragments generated in step iii) function asprimers for all nucleic acid polymerases capable of extending standardnucleic acid primers.

The use of a labelled precursor nucleotide or dideoxy terminatornucleotide in any of the extension reactions facilitates detection ofthe extended extendible fragment. Direct DNA staining methods such assilver or ethidium bromide staining facilitate detection of allextension products after size separation based on electrophoreticmobility.

The ability of the extendible fragments to function in a subsequentextension reaction using a template nucleic acid and normal or dideoxyterminator nucleotides (a nucleotide that prohibits further extension ofa primer on a template once incorporated) generates a ladder offragments allowing determination of the location of the total number ofpositions of the modified precursor nucleotide in one or both strands ofthe amplified target nucleic acid. The presence of a sequence variationor mutation results in the appearance or disappearance of a cleavagefragment as judged by comparison with the known DNA sequence of theamplified molecule. Size analysis of the fragments allows the preciselocation and sequence of a mutation in the target nucleic acid sample tobe determined. Therefore, if a sequence variation occurs such that anadditional site of modified precursor nucleotide incorporation isgenerated, an additional cleavage fragment will be observed uponanalysis of the ladder of cleavage products. If a sequence variationoccurs such that a site of modified precursor nucleotide incorporationis lost, the corresponding cleavage fragment will not be observed uponanalysis of the ladder of cleavage products.

The template of choice in this case will be the originally intact orglycosylase cleaved amplified nucleic acid. In cases where glycosylasecleaved amplified nucleic acid is used it can be processed to removeresidual moieties downstream of the extendible fragment which prohibitextension of the extendible fragment by a nucleic acid polymerase.Specifically the template DNA can be treated so that a residual 5′deoxyribose moiety is removed. This is achieved by incubation of thetemplate DNA with a 5′ deoxyribophosphodiesterase such as E. coli RecJendonuclease or formamidopyrimidine DNA glycosylase. Other naturallyoccurring or enzymatically or chemically synthesised template nucleicacids which fully or partially hybridise to the extendible fragment canalso be designed and/or selected as a template nucleic acid to determinethe ability of the extendible fragment to function as a primer.

The ability of the extendible fragments to function in a subsequentextension reaction using a template nucleic acid and a combination ofnon-labelled and labelled normal or dideoxy terminator nucleotidespermits detection of sequence variations and mutations. Extension of theextendible fragments on the template nucleic acid from which they werederived and which is heterozygous for the sequence variation, using alabelled dideoxy terminator nucleotide having base pairing propertiesdifferent to those of the modified precursor nucleotide with anon-labelled dideoxy terminator nucleotide having the same base pairingproperties as the modified precursor nucleotide allows detection of thevariant or mutant loci alone whereas non variant loci are not detected.This aspect of the invention is particularly advantageous as detectionof sequence variations alone permits very high throughput mutationscanning and detection, and allows fingerprinting of nucleic acids basedon their sequence variations.

It will be appreciated that amplification of any target DNA, which isheterozygous for a mutation or polymorphism generates four distinctduplex DNAs, i.e. (taking as an example a G to A mutation at position Xin a DNA sequence), one quarter will be homoduplex with a GC base pairat position X, one quarter will be homoduplex with an AT base pair atposition X, one quarter will be heteroduplex with a GT base pair atposition X, and one quarter will be heteroduplex with a AC base pair atposition X. Similarly, heteroduplex DNA can be generated readily bydenaturing and reannealing two homoduplex DNAs bearing a sequencedifference(s).

Thus, the nature of the sequence of the 3′end of the extendiblefragments can be determined by their ability to function as primers in asubsequent extension reaction using a template nucleic acid.Essentially, such determination is based on the ability of the 3′end ofthe extendible fragment to hybridise to a selected template underselected conditions. Following partial or complete hybridisation theextendible fragment may be extended using a nucleic acid polymerase andnucleic acid precursors or selected combinations of same as hereindescribed. It will be appreciated that multiple possibilities exist forthe selection of template molecules. Nonetheless, the extension of theextendible fragment is a measure of its hybridisation to or lack ofhybridisation to a selected template molecule and thus the determinationof the nature of the sequence of the 3′end of the extendible fragmentsis made on this basis, since this 3′ sequence is indicative of thesequence of the original target nucleic acid.

Typically, the template molecule is selected so that it bears partial orfull sequence complementarity to the upstream fragment. The upstreamfragment may be extended one or more nucleotides on the templatemolecule using a nucleic acid polymerase and nucleic acid precursors ora combination of same or dideoxy terminator nucleotides or a combinationof normal nucleic acid precursors and dideoxy terminator nucleotides.

The extension of step iv) can be achieved by means of an amplificationreaction using said extendible DNA fragment.

Alternatively, the extension of step iv) is achieved by means of anamplification reaction including a primer in addition to using saidextendible DNA fragment.

Repeated extension of an upstream fragment on a template nucleic acid incombination with a second flanking primer which can be extended on thecopy of the template permits amplification of the template nucleic acid.Such amplified products can be readily detected by standard nucleic acidstaining methods such as ethidium bromide after resolution byelectrophoresis.

Alternatively, the template molecule may be selected so that it can beextended using the upstream fragment as a template and so that theextension is based on hybridisation to the 3′ end of the upstreamfragment.

Also, the upstream fragment may be analysed based on its ability tofunction in a 5′ nuclease assay. During extension of the upstreamfragment by a polymerase with 5′ to 3′ nuclease activity, the 5′ to 3′nuclease activity degrades a downstream reporter molecule annealled tothe same template strand as said upstream fragment.

A further possibility is for the upstream fragment to be extended on asynthetic template which contains reporter and quencher labels, thencleavage of the resulting double stranded DNA will release the reporterfrom the quencher and a signal will be detected. Typically such cleavagewill be carried out by an enzyme which recognises the double strandedDNA molecule. Typically such an enzyme will be a restriction enzyme.

Furthermore, the 3′ terminal sequence of the upstream fragment may bedetermined on the basis of its hybridisation to other nucleic acidmolecules.

A further possibility is that the products extended or amplified usingupstream fragments may be detected on the basis of their filtrationand/or precipitation properties.

When analysing extension and incorporation of nucleotides in step iv)where the upstream fragments are used in an extension reaction, it isimportant to verify that any extension observed is specifically due toextension of the upstream fragments and not due to extension of theinitial primers which were unused during the initial amplification, ifsuch is used in step i), or extension of possible non-specific upstreamfragments which may be generated by the non-specific breakage or damageof the DNA during previous steps of the procedure. To overcome this‘noise’, the DNA, prior to glycosylase mediated treatment, can betreated with a single strand specific DNA nuclease, for example,Exonuclease I, which will degrade the unused primers and non-specificsingle stranded DNA and can be subsequently heat inactivated. The DNAcan be treated with a 3′ AP endonuclease/lyase which will cut the DNAand primers at any preexisting AP sites, generated specifically orgenerated through damage of the DNA. The 3′ AP endonuclease/lyase issubsequently removed from the reaction. Because the endonuclease/lyasecuts at the 3′ side, the resulting contaminating upstream fragments arenot extendible and will not interfere with the extension of thesubsequently generated upstream fragments. In addition to thesetreatments, a control reaction to check for non-specific extensions canbe carried out. Thus, in step iii) of the method, the AP sites can becut with a 3′ AP endonuclease/lyase, thereby generating non-extendibleupstream fragments. If however, extension and incorporation ofnucleotides is observed in the subsequent step iv) then one can measureor determine the level of non-specific extension obtained during theprocedure.

Similarly it is important to ensure that the incorporation ofnucleotides, labelled or unlabelled, in step iv) is due to theincorporation of those nucleotides supplied during step iv) and notthose from any previous step. This is especially important when thereaction involves incorporation of dideoxynucleotides. Prior to orsubsequent to cleaving the DNA at abasic sites and generating upstreamfragments, the reaction can then be treated with a phosphatase whichdigests all unincorporated nucleotides present in the reaction, e.g.shrimp alkaline phosphatase, which can subsequently be heat inactivated.

In a further embodiment of the invention, the enzyme used in step iv) isa ligase which can be incubated with the extendible upstream fragment inthe presence of a reporter oligonucleotide.

The reporter oligonucleotide may be partially degenerate.

The method according to the invention can be used inter alia to detect aknown or unknown mutation and to detect differences and similarities ingenomes. These aspects of the invention are illustrated further below.

The method according to the invention provides in one aspect a means ofgenerating random primers in a simple easy manner. Essentially,introduction of a modified base into an amplified DNA product followedby glycosylase mediated cleavage and subsequent treatment of thecleavage products so that they can be extended by a nucleic acidpolymerase provides a rapid means of generating random primers.Subsequent use of such primers, i.e., the extendible upstream fragments,in random amplification of target nucleic acids allows amplification ofdiscrete DNA molecules from the target nucleic acids thus permittingcharacterisation of the nucleic acid based on similarities anddifferences of the amplified products. Since these primers areessentially derived from said target nucleic acid, specifically their 3′ends, they are better primers for the subsequent random amplificationanalysis of said target nucleic acid and the amplification is morespecific. Many discrete DNA products are generated during randomamplification of nucleic acids. A discrete DNA product can be separatedfrom other products on the basis of size. The method according to theinvention permits the generation of primers from all or part of theseparated product. Use of an initial primer in the random amplificationof a nucleic acid that permits immobilisation of the separated productallows the isolation of the upper and lower primers extended to thefirst point of glycosylase mediated cleavage. The 3′ end of suchupstream fragments are derived from the target nucleic acid and thuspermit more specific amplification of the target nucleic acid or relatednucleic acids. The 3′ end of such upstream fragments may be short orlong. By short 3′ ends herein is meant one to three nucleotides whereasby long 3′ ends herein is meant greater than three nucleotides. Longer3′ ends on such upstream fragments are more desirable as they allowhighly specific amplification of a target nucleic acid sequence.Upstream fragments generated with longer 3′ ends may be selected bysizing methods. Alternatively, the initial amplification primers can bedesigned so that they promote binding of a protein that protects asection of the region 3′ of the initial extended primer. Thus such aregion is refractory to glycosylase mediated cleavage due to protectionby the protein and inclusion of such a primer design and protein allowsgeneration of upstream fragments with longer 3′ ends.

This embodiment of the invention provides a rapid and simple method forgeneration of random and specific primers for nucleic acid amplificationwithout prior knowledge of the nucleic acid sequence.

It will be appreciated that the random amplification of nucleic acidusing arbitrarily chosen primers is a method already known for detectingsimilarities and differences between genomes. Such random amplificationis based on the annealing of arbitrarily chosen primers to targetsamples followed by multiple rounds of enzymatic amplification wherebythe primers are extended using the selected genomic DNA or cDNA astemplate. Using such primers in such a method results in theamplification of discrete DNA molecules. Analysis of such moleculesallows the investigation of similarities and differences betweendifferent samples. Typically many different random primers are chosenfor investigation of a genome or cDNA and such primers are synthesisedchemically and are designed in a random fashion with the assumption thatthey will hybridise to the target nucleic acid in the amplificationprocess. The method according to the invention allows the easy and rapidgeneration of primers from a target nucleic acid which can subsequentlybe used for random amplification of the same or different target nucleicacid.

In addition to extension by a polymerase reaction, as indicated above,the upstream fragments in step iv) can also be extended by ligation ofanother single stranded DNA molecule which results in extended upstreamfragments greater in size than the initial upstream fragments generatedby glycosylase mediated cleavage. The DNA molecule to which the upstreamfragment is ligated is termed the reporter oligonucleotide herein andthis can vary in length. Ligation of the upstream fragments to thereporter oligonucleotide is dependent on both DNA molecules (theupstream fragment and reporter oligonucleotide) annealing to a templatemolecule at adjacent sites so that the termini of the upstream fragmentand reporter oligonucleotide are juxtaposed. This means that the 3′terminal base of the upstream fragment is juxtaposed to the 5′ terminalbase of the reporter oligonucleotide. The reporter oligonucleotide istypically a synthetic oligonucleotide, but can also be any other type ofDNA molecule or RNA molecule.

The template molecule is selected so that it bears partial or fullsequence complementarity to the upstream fragment and reporteroligonucleotide. The template of choice can be the originally intact orglycosylase cleaved amplified nucleic acid. In addition the template canbe a single stranded DNA molecule, e.g. synthetic oligonucleotide, whichcan vary in length and which will allow complementary annealing of theupstream fragment(s) and reporter oligonucleotide(s). The template DNAcan be single stranded or double stranded in nature. Double stranded DNAacting as template consists of the template strand and the complementarystrand. Double stranded DNA must first be denatured and then allowed toreanneal in the presence of the upstream fragment and reporteroligonucleotide. The upstream fragment and reporter oligonucleotide thencompete with the complementary strand in annealing to the templatestrand of the double stranded template molecule.

Various DNA ligases and RNA ligases can be used to extend the 3′terminus of the extendible fragment by ligation of a reporteroligonucleotide on a template nucleic acid. DNA ligases from manysources, including those isolated from phage, e.g. T4 DNA ligase, andmesophilic and thermophilic bacteria, can be used to ligate the reporteroligonucleotide to the extendible fragment. Thermophilic DNA ligases canbe used in repeated cyclic ligations of the extendible fragments toreporter oligonucleotides.

As discussed above, the method according to the invention involves theproduction of an upstream fragment which has a 3′ hydroxyl group. Thisis an essential requirement for extension of the molecule by addition ofnucleotides by a polymerase and also for extension by ligation of areporter oligonucleotide by a ligase. In addition to a 3′ hydroxyl groupon the upstream fragment, the reporter oligonucleotide is required tohave a 5′ phosphate group for ligation to occur. Ligation, in addition,allows detection of the downstream fragment if desired. The downstreamfragment is the remainder of the DNA strand from which the upstreamfragment was cleaved. Here, the reporter oligonucleotide is required tohave a 3′ hydroxyl terminus and the downstream fragment to have a 5′terminal phosphate group.

Since glycosylase mediated cleavage may generate several differentupstream fragments in an individual reaction due to the presence ofnormal and mutant alleles in the target nucleic acid or due to therandom introduction of modified bases, the extension by ligationreaction may contain several different upstream fragments in addition toseveral different reporter oligonucleotides and template nucleic acids.In addition several different nucleic acids may be characterisedsimultaneously since an individual reporter oligonucleotide and/ortemplate nucleic acid may be used for the characterisation of eachindividual nucleic acid under investigation. The extended upstreamfragment can be detected by any of several means including sizeanalysis, hybridisation and amplification. In addition, the DNA moleculeresulting from ligation can be further amplified in a polymerase chainreaction.

The reporter oligonucleotide, upstream fragment or template nucleic acidmay be labelled. For example a fluorescent or radioactive label may beused in addition to a biotin or digoxigenin label. A useful label on thereporter oligonucleotide is a 5′ terminal radioactive phosphate, i.e.³²P or ³³P. This radioactive phosphate serves as a label with which todetect the DNA and also as the necessary 5′ phosphate on the reportermolecule. A biotin label on the reporter oligonucleotide, upstreamfragment or template nucleic acid will serve to immobilise the extendedupstream fragment directly, or via hybridisation, to a solid support.Immobilisation, in combination with multiple different ligationextensions will serve to produce a very efficient and high throughputsystem for characterisation of DNA molecules.

The invention, using extension by ligation, may also be used to identifyunknown sequence changes in nucleic acid by its ability to identify amutant upstream fragment in a mixture of normal upstream fragments. Apartially degenerate reporter oligonucleotide may be used in theligation reaction. Selective ligation of an upstream fragment arisingfrom cleavage of a DNA molecule at a mutation site can also be achievedby using a degenerate reporter oligonucleotide where the 5′ terminus iscomplementary to the normal allele. In addition the invention can beused to investigate all CpG dinucleotide sites within a DNA fragment byusing a fully complementary reporter oligonucleotide designed to annealat each CpG site. The length of any resulting ligation product willindicate the CpG site which has been mutated.

Suitably, any extended fragments resulting from step iv) are detected byhybridisation.

The method according to the invention offers significant advantages overexisting methods in that

a) it allows detection of the similarities or the differences, or thesimilarities and differences between nucleic acid samples. Inparticular, it allows the detection of same at large numbers of multipledifferent loci in a nucleic acid. While it is possible to use othermethods to achieve this end point, this method offers the advantage of asingle process which can readily be scaled up allowing rapid and easycharacterisation of nucleic acid molecules.

b) It will be appreciated that amplification of nucleic acids is acommon method for characterising and detecting nucleic acids.Amplification is dependent on the use of primers which are extended inthe amplification process. In addition, the method according to theinvention allows the generation of high specificity primers foramplification of nucleic acids without the necessity of prior knowledgeof any of the sequence of the nucleic acid. Thus, the present method hasa high utility for the characterisation of nucleic acids throughanalysis of amplification products generated therefrom. Theamplification approaches described for characterising nucleic acidsaccording to the invention allow characterisation of nucleic acids in away that was not possible prior to the present invention.

c) in the field, there is a need for simplified methods for thedetection of specific mutations at candidate loci. The method accordingto the invention offers such a simplified method for the detection ofsuch mutations. In particular, the upstream fragment generated fromsequence variation loci in nucleic acids allow the analysis of such lociusing many different analytical approaches and allow accurate andsimplified detection of sequence variations at such loci.

d) the method according to the invention offers a more reproduciblemethod for characterising nucleic acids by random amplification and isless susceptible to error.

The method according to the invention can also be used to analyse theCpG content of DNA by detecting C to T transitions in DNA.

Upon analysis of mutation data and mutation spectra that have beengenerated over many years of mutation research, a definite hotspot formutations in all organisms which contain 5-methylcytosine in theirgenomic DNA e.g. humans, has been identified, namely, mutations at CpGdinucleotides. CpG dinucleotides are a site for cytosine methylation inhuman cells and have been implicated in many structural and regulatoryroles in genome organisation and gene expression, respectively. Cytosinein DNA is normally susceptible to a low but measurable level ofdeamination to form uracil, an event which is mutagenic if not repaired.However, upon methylation at the 5′ site, the cytosine ring now is evenmore susceptible to spontaneous deamination. Therefore, the5-methylcytosine residue is deaminated to become thymine. Followingdeamination, the CpG dinucleotide becomes a TpG dinucleotide. Therefore,since 5-methylcytosine occurs only at a CpG, and the major cause ofmutation at this site is due to deamination of the 5-methylcytosine,mutated CpG sequences appear in DNA as TpG dinucleotide sequences whichrepresents a classic C to T transition mutation. Since CpG has beendemonstrated to be such a hotspot for mutation in human genetic studies,a rapid screen for enhanced detection of mutations at CpG dinucleotideswithin the test fragment of DNA is highly advantageous.

To enhance the detection of mutations at CpG sequences, one of thefollowing procedures can be carried out subsequent to amplification ofthe target DNA followed by cleavage to generate the upstream fragments.

Step iv) is carried out in the presence of a polymerase,dideoxyTTP(ddTTP), labelled dideoxy CTP(ddCTP) and wild type DNA astemplate, the upstream fragments are extended by incorporation ofdideoxynucleotides, but only sites where C has mutated to T, includingsites where CpG has mutated to TpG will become labelled following theincorporation of the labelled ddCTP. Since the ddCTP is a chainterminator nucleotide, the DNA will not be extended beyond this point.Therefore the DNA will be extended at mutated CpG sequences and willalso be labelled and detectable.

Step iv) is carried out in the presence of a polymerase, wild type DNAas template, dTTP and labelled dideoxyGTP (ddGTP), the upstreamfragments are extended by incorporation of dTMP, but only sites wherethe T is followed by G, i.e. TpG sequences will become labelledfollowing the incorporation of the labelled ddGTP. Since the ddGTP is achain terminator nucleotide, the DNA will not be extended beyond thispoint. Therefore the DNA is now extended at all TpG sequences and isalso labelled and detectable. Since TpG dinucleotides occur naturally inDNA as well as arising from CpG deamination, wild type DNA will show acharacteristic banding pattern (normally only a few bands will be seen).The label can be e.g. radioactive ³³P, fluorescent, or biotin labelledin a manner known per se.

Step iv) is carried out in the presence of polymerase, dUTP, dGTP, dATP,labelled dCTP and wild type DNA as template, the upstream fragments areextended by incorporation of deoxynucleotides and become labelled. Theextended upstream fragments are subsequently cleaved by uracil DNAglycosylase and abasic site cleavage agent. Only that upstream fragmentwhich was generated by cleavage at the mutation site will remainextended and labelled. This procedure will detect all mutationsgenerating a T incorporation site including C to T mutations at CpGsites. This procedure has the advantage that step iv) utilises theincorporation of deoxynucleotides which are incorporated moreefficiently than dideoxynucleotides by all polymerases.

In addition, in heterozygous samples, the mutated CpG will give rise toT/G mismatches (or U/G after introduction of dUTP as modifiednucleotide). The thymine DNA glycosylase can be used to specificallycleave at these T/G or U/G mismatches at a mutated CpG sequence.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram of an AP site generated during one step of themethod according to the invention and of a number of ways in which theDNA at the abasic site can be cleaved so as to generate an upstream DNAfragment that can be extended;

FIG. 2 is a schematic representation of the method according to theinvention as described in Example 1;

FIG. 3 is a schematic representation of the method according to theinvention as described in Example 2 where a labelled ddTTP was used in alinear amplification reaction following generation of an upstreamextendible fragment;

FIG. 4 is a schematic representation of the method according to theinvention as described in Example 2 where a labelled ddCTP was used in alinear amplification reaction following generation of an upstreamextendible fragment;

FIG. 5 is a schematic representation of the method according to theinvention as described in Example 3;

FIGS. 6A–6D is a schematic representation of the extension productsobtained in Example 3 following electrophoresis and autoradiography andanalysis of the autoradiographs; and

FIGS. 7A–7D is a schematic representation of the ligation reactionscarried out in Example 4 on the upstream fragments and the productsthereby obtained.

FIG. 1 depicts a single strand of DNA in 5′ to 3′ orientation. Theposition of two bases is shown, i.e. base 1 and base 3. Base 2 has beenremoved and an AP site exists at this position. The vertical linesdenote the ribose ring which is attached to the base. The diagonal lineswith ‘P’ in a circle refer to the phosphodiester linkages linking eachribose. The DNA can be cut at the 5′ side or 3′ side of an AP site asshown by arrows. If the DNA is cleaved at the 3′ side of the AP site,i.e. position Z, then the upstream fragment (containing base 1) has adeoxyribose phosphate moiety at its 3′ terminus as shown in box C.Cleavage of the DNA at the 3′ side of the AP site can be achieved bytreatment with a 3′ AP endonuclease/lyase or heat treatment ashereinabove described.

The AP site can also be cut in two different ways on the 5′ side of theAP site, i.e. at positions X and Y. Cleavage at position X results in aOH group at the 3′ terminus on the upstream fragment, as shown in box A.Cleavage at position X can be achieved by treatment with a 5′ APendonuclease as hereinabove described. Cleavage at position Y results ina phosphate group at the 3′ terminus of the upstream fragment, as shownin box B. Cleavage at position Y can be achieved by treatment with heatand alkali as hereinabove described.

The invention will be further illustrated by the accompanying Examples.

MODES FOR CARRYING OUT THE INVENTION Example 1

The method according to the invention was used to demonstrate theproduction of an extended DNA fragment by extension from the 3′OH groupof an upstream fragment, which had been generated by cleavage of uracilcontaining DNA at the site of incorporation of the modified nucleotide.Target nucleic acid was a region of the RYR1 gene (952 to 1044)amplified from human cDNA by using upper (952 to 972) and lower (1024 to1044) primers to generate a double stranded DNA, 93 bp in length.(Nucleotide numbers refer to the sequence of the RYR1 gene)

FIG. 2 is a schematic diagram of the target nucleic acid and the upperand lower primers (primers contain standard bases, G, A, T and C). Sixpmoles of the lower primer was end labelled by incubation with 1 unit ofT4 Polynucleotide Kinase (commercially available from New EnglandBiolabs), 70 mM Tris-HCl (pH7.6), 10 mM MgCl₂, 5 mM dithiothreitol and 1μCi γ³²P ATP (3000 Ci/mmol) for 30 min at 37° C. The target nucleic acidsample was amplified by PCR in a reaction mix containing target nucleicacid, 0.2 mM dATP, dCTP, dGTP and dUTP, 6 pmoles of ³²P labelled lowerprimer and non-labelled upper primer in a total volume of 19 μl. Thereaction mix was then overlaid with an equal volume of mineral oil and ahot start PCR was performed whereby the reaction mix was heated to 94°C. for 5 min prior to addition of 1 unit of Taq polymerase (availablefrom Promega) (bringing the total volume to 20 μl). 30 cycles ofdenaturation, annealing and extension were carried out in athermocycler. The reaction mixture bearing the amplified target nucleicacid was then treated with exonuclease I (available from Amersham LifeSciences) to digest the primers not extended in the amplification stepand shrimp alkaline phosphatase (SAP) (available from BoehringerMannheim) to digest the dNTPs not incorporated during the amplificationstep. This was achieved by incubating 10 μl of the PCR reaction with 0.5units of exonuclease I and 1 unit of SAP at 37° C. for 30 min. Exo I andSAP were subsequently heat inactivated by incubating the reaction at 80°C. for 15 min.

Uracil DNA-glycosylase (available from New England Biolabs) (0.5 units)was then added and the incubation continued at 37° C. for 30 min.Following treatment with uracil DNA-glycosylase, the abasic sitesgenerated in the amplified product were cleaved to completion by addingNaOH to a final concentration of 0.05M and heating the mixture for 15min at 95° C. The digested DNA was then precipitated by adding 10%volume 3M sodium acetate and 2 volumes of ethanol. The pellet wasresuspended in 5 μl water. The digested DNA was then treated with 0.5units of T4 polynucleotide kinase (PNK) which removes the phosphategroup from the 3′ terminii.

A linear amplification reaction was then carried out using the productsof the above cleavage reaction, the fragment of interest being thelabelled upstream extendible fragment, during which, the upstreamextendible fragment is extended by a thermostable DNA polymerase in acycling reaction in a total volume of 10 μl . The template for thisreaction is amplified target nucleic acid (952 to 1044 of RYR1 gene)which is free from primers due to pretreatment with ExoI.

An equal volume of formamide loading dye (90% formamide, 0.025%bromophenol blue, 0.025% xylene cylanol) was added to the sample whichwas then heated at 85° C. for 5 min. The sample was then loaded onto a20% denaturing (7M urea) polyacrylamide gel and electrophoresis wascarried out for 3–4 hours at 60 W for size analysis of the extensionproducts. Following electrophoresis, autoradiography was carried out byexposing the gel directly to X-ray photographic film for 12 hrs at −70°C.

Analysis of the autoradiograph, where the lower primer was labelled,showed a product of 93 nucleotides in length. This product was notobserved if the T4 polynucleotide kinase treatment or the linearamplification reaction was not included in the above procedure.

Example 2

The method according to the invention was used to detect the presence ofa G to A mutation at position 1021 in the human RYR1 gene. cDNA from anormal individual and from an individual affected with MalignantHyperthermia was amplified by using upper (952 to 972) and lower (1204to 1224) primers to generate a 273 bp double stranded DNA fragment (952to 1224)(As in the case of Example 1 nucleotide numbers refer to thesequence of the RYR1 gene).

FIGS. 3 and 4 are schematic diagrams of the target nucleic acid and theupper and lower primers (primers contain standard bases, G, A, T and C).The target nucleic acid sample was amplified by PCR in a reaction mixcontaining target nucleic acid, 0.2 mM dATP, dCTP, dGTP and 0.19 mM dTTPand 0.01 mM dUTP, and 6 pmoles of upper and lower primers in a totalvolume of 19 μl. The reaction mix was then overlaid with an equal volumeof mineral oil and a hot start PCR was performed whereby the reactionmix was heated to 94° C. for 5 min prior to addition of 1 unit of Taqpolymerase (bringing the total volume to 20 μl ). 30 cycles ofdenaturation, annealing and extension were carried out in athermocycler. The reaction mixture bearing the amplified target nucleicacid was then treated with exonuclease I to digest the primers notextended in the amplification step and shrimp alkaline phosphatase (SAP)to digest the dNTPs not incorporated during the amplification step. Thiswas achieved by incubating 10 μl of the PCR reaction with 0.5 units ofexonuclease I and 1 unit of SAP at 37° C. for 30 min. Exo I and SAP weresubsequently heat inactivated by incubating the reaction at 80° C. for15 min.

Uracil DNA-glycosylase (0.5 units) was then added and the incubationcontinued at 37° C. for 30 min. Following treatment with uracilDNA-glycosylase, the abasic sites generated in the amplifiedproduct-were cleaved to completion by adding NaOH to a finalconcentration of 0.05M and heating the mixture for 15 min at 95° C. Thedigested DNA was then precipitated by adding 10% volume 3M sodiumacetate and 2 volumes of ethanol. The pellet was resuspended in 5 μlwater. The digested DNA was then treated with 0.5 units of T4Polynucleotide Kinase which removes the phosphate group from the 3′terminii.

A linear amplification reaction was then carried out using the productsof the above cleavage reaction, i.e. the various extendible upstreamfragments, during which, the upstream fragment is extended by athermostable DNA polymerase (i.e. Thermosequenase (available fromAmersham Life Sciences)) in a cycling reaction in a total volume of 10μl. The template for this reaction is amplified fragment of normal cDNA(952 to 1224 of RYR1 gene) which is free from unlabelled primers due topretreatment with ExoI. The extension reaction is carried out in thepresence of 1 mM of three of the dideoxy terminator nucleotides (ddNTP)and 0.02 mM of a ³³P-labelled ddNTP.

An equal volume of formamide loading dye (90% formamide, 0.025%bromophenol blue, 0.025% xylene cylanol) was added to the sample whichwas then heated at 85° C. for 5 min. The sample was then loaded onto a6% denaturing (7M urea) polyacrylamide gel and electrophoresis wascarried out for 3–4 hours at 60 W for size analysis of the extensionproducts. Following electrophoresis, autoradiography was carried out byexposing the gel directly to X-ray photographic film for 12 hrs at −70°C.

Analysis of the autoradiograph, where ddTTP was the labelled ddNTP (FIG.3), showed a ladder of labelled fragments corresponding to the distancefrom the 5′ end of the primers to the site of a dUMP incorporation (Onlyextension of the lower strand is shown). These sites corresponded to thewild type T pattern of the DNA. When mutant target nucleic acid was usedto generate the extension primers, an additional band was observed inthe T pattern of bands (203 nucleotides). Analysis of theautoradiograph, where ddCTP was labelled (FIG. 4) showed no labelledbands when amplified normal target nucleic acid was the source ofupstream fragments, however, when amplified mutant target nucleic acidwas used, analysis showed just one band. The size of the labelledfragment corresponds to the distance between the 5′ end of the lowerprimer and the mutation site (i.e. 203 nucleotides) therebydemonstrating the presence of the G to A mutation at position 1021 ofthe RYR1 gene in that individual.

Example 3

The method according to the invention was used to detect the presence ofa G to A mutation at position 6411 (codon 12) in the human Ki-ras gene.Genomic DNA from normal tissue and tumour tissue of an individual withcolon cancer was amplified by using upper (6390 to 6409) and lower (6417to 6443) primers to generate a 54 bp double stranded DNA fragment (6390to 6443)(Nucleotide numbers refer to the genomic sequence of the Ki-rasgene including introns).

FIG. 5 is a schematic diagram of the target nucleic acid and the upperand lower primers (primers contain standard bases, G, A, T and C). Thetarget nucleic acid sample was amplified by PCR in a reaction mixcontaining target nucleic acid, 0.2 mM dATP, dCTP, dGTP and dUTP and 6pmoles of upper and lower primers in a total volume of 19 μl. Thereaction mix was then overlaid with an equal volume of mineral oil and ahot start PCR was performed whereby the reaction mix was heated to 94°C. for 5 min prior to addition of 1 unit of Taq polymerase (bringing thetotal volume to 20 μl). 30 cycles of denaturation, annealing andextension were carried out in a thermocycler. The reaction mixturebearing the amplified target nucleic acid was then treated withExonuclease I (Exo I) to digest the primers not extended in theamplification step and Shrimp Alkaline Phosphatase (SAP) to digest thedNTPs not incorporated during the amplification step. This was achievedby incubating 20 μl of the PCR reaction with 0.5 units of Exo I and 1unit of SAP at 37° C. for 30 min. Exo I and SAP were subsequently heatinactivated by incubating the reaction at 80° C. for 15 min. UracilDNA-glycosylase (0.5 units) and Endonuclease IV (1 unit) were then addedand the incubation continued at 37° C. for 30 min to allow completeexcision of all the uracils present in the amplified DNA and cleavage ofthe resulting abasic sites to completion.

This cleavage resulted in the upstream fragments having a 3′ hydroxylgroup at their 3′ terminus which can be extended by action of a DNApolymerase.

Extension reactions were then carried out in a reaction mixture (10 μl)containing the products of the above cleavage reaction, i.e. theextendible upstream fragments (2 μl of cleavage reaction, approx. 1 pmolof extendible fragments), and 100 fmol of a synthetic templateoligonucleotide, 0.2 mM dATP, dTTP, and dGTP, 0.02 mM dCTP, 1 μCiα³²PdCTP, 6 pmol reverse primer and 1 unit Taq DNA polymerase. Thereaction was carried out for 40 cycles of denaturation, annealing andextension.

An equal volume of formamide loading dye (90% formamide, 0.025%Bromophenol blue, 0.025% Xylene cylanol) was added to the sample whichwas then heated at 85° C. for 5 min. The sample was then loaded onto adenaturing (7M urea) polyacrylamide gel and electrophoresis was carriedout for 3–4 hours at 60 W for size analysis of the extension products.Following electrophoresis, autoradiography was carried out by exposingthe gel directly to X-ray photographic film for 12 hrs at −20° C.

Analysis of DNA from normal tissue results in the generation of a 37nucleotide extendible fragment following glycosylase mediated cleavageusing the above mentioned upper and lower primers (FIG. 5). Similaranalysis of DNA from tumour tissue results in the generation of a 32nucleotide extendible fragment (FIG. 5). Analysis of the autoradiographshowed a 66 nucleotide band following analysis of DNA from normal tissueand using template oligonucleotide 1 (FIG. 6A). This band was notobserved when template oligonucleotide 2 was used in the above analysis(FIG. 6B). Analysis of the autoradiograph showed a 66 nucleotide bandfollowing analysis of DNA from tumour tissue and using templateoligonucleotide 2 (FIG. 6C). This band was not observed when templateoligonucleotide 1 was used in the above analysis (FIG. 6D). Thereforethe presence of a mutation at codon 12 of the Ki-ras gene was determinedby the ability of the upstream fragment to be extended on a mutanttemplate oligonucleotide whereas the upstream fragment was not extendedon a normal template oligonucleotide and vice versa for the absence ofthe mutation.

Example 4

Example 3 was repeated to the stage where the abasic sites had beencleaved to completion. This cleavage resulted in the upstream fragmentshaving a 3′ hydroxyl group at their 3′ terminus as before.

Reporter oligonucleotides 1 and 2 (FIG. 7)(complementary to nucleotides6397 to 6406 (5′ TCCAACTACC3′ No. 1 (SEQ ID NO. 28)) and nucleotides6402 to 6411 (5′CCAGCTCCAA3′, No. 2 (SEQ ID NO. 30)) of the Ki-ras generespectively) were 5′ endlabelled using γ³²PATP and T4 polynucleotidekinase. This served to label the resulting ligated fragment and alsoprovided a 5′ terminus phosphate on the reporter oligonucleotide whichis required for any possible ligation.

Ligation reactions (16° C. for 60 min) were then carried out using theproducts of the above cleavage reaction, i.e. the extendible upstreamfragments (5 μl of cleavage reaction, approx. 2 pmol of extendiblefragments), 4 pmol of labelled reporter oligonucleotide and 2 pmol of atemplate oligonucleotide 1 or 2 (5′GGTAGTTGGAGCTGGTGGCG3′ (SEQ ID No.27) (nuc 6397 to 6416) or 5′TTGGAGCTGGTGGCGTA GGC3′ (SEQ ID No. 31) (nuc6402 to 6421) respectively) (FIG. 7) and 1 unit T4 DNA ligase, duringwhich, the upstream fragment is extended in length by the ligation of areporter oligonucleotide in a 20 μl reaction.

An equal volume of formamide loading dye (90% formamide, 0.025%Bromophenol blue, 0.025% Xylene cylanol) was added to the sample whichwas then heated at 85° C. for 5 min. The sample was then loaded onto adenaturing (7M urea) polyacrylamide gel and electrophoresis was carriedout for 3–4 hours at 60 W for size analysis of the extension products.Following electrophoresis, autoradiography was carried out by exposingthe gel directly to X-ray photographic film for 3 hrs at −20° C.

Analysis of DNA from normal tissue results in the generation of a 37nucleotide extendible fragment following glycosylase mediated cleavageusing the above mentioned upper and lower primers (FIG. 5). Similaranalysis of DNA from tumour tissue results in the generation of a 32nucleotide extendible fragment (FIG. 5). Analysis of the autoradiographshowed a 47 nucleotide band following analysis of DNA from normal tissueand using reporter oligonucleotide 1 and template oligonucleotide 1(FIG. 7A). This band was not observed when reporter oligonucleotide 2was used in the above analysis (FIG. 7B). Analysis of the autoradiographshowed a 42 nucleotide band following analysis of DNA from tumour tissueand using reporter oligonucleotide 2 and template oligonucleotide 2(FIG. 7C). This band was not observed when reporter oligonucleotide 1was used in the above analysis (FIG. 7D). Therefore the presence of amutation at codon 12 of the Ki-ras gene was determined by the presenceof a 42 nucleotide band whereas the presence of the normal allele wasdetermined by the presence of a 47 nucleotide band. Samples containingnormal and tumour DNA yielded both 42 and 47 nucleotide bands.

The above analysis was also carried out where the lower primer was 5′endlabelled with ³²P. This required that the reporter oligonucleotidesbe phosphorylated using unlabelled ATP as phosphate donor. The resultswere similar to those observed above in that a 42n band demonstrated thepresence of the mutant Ki-ras gene (codon 12) whereas a 47n banddemonstrated the absence of the mutation at codon 12 of the Ki-ras gene.In addition the above analysis was also carried out using the initiallyamplified Ki-ras gene fragment from normal or mutant sample as templateduring the ligation reaction. Normal amplified product was used insteadof template oligonucleotide 1, whereas mutant amplified product was usedinstead of template oligonucleotide 2. Again the same results wereobserved as described above.

As indicated above, the method according to the invention has numerousadvantages over known methods, especially the method of WO 97/03210.After the glycosylase mediated cleavage in the case of WO 97/03210(which can be carried out in a number of ways and which yields a numberof different 3′ termini), the resulting DNA fragments are not furtherprocessed and are analysed directly. In the present invention, theglycosylase mediated cleavage step is followed by a step that permitsextension of the 3′ termini generated by glycosylase mediated cleavage.

A major advantage of the present invention, which is not possible in thecase of the prior art methods, is that the present invention allowsdetection of sequence differences such as mutations and polymorphismsbetween nucleic acid molecules without detecting sequence similarities.As indicated above, it is not possible with the method of WO 97/03210 todetect sequence differences between nucleic acid molecules withoutdetecting sequence similarities. This is a limitation of the method ofWO 97/03210 as multiple samples cannot be combined for simultaneousanalysis. The present invention also allows for the analysis of multiplegenes or gene segments simultaneous analysis. Furthermore, the presentinvention allows for the generation of specific primers foramplification of nucleic acids without the necessity of having priorknowledge of the sequence of the nucleic acid. This is not possible withthe known methods. In addition, the present invention permits thegeneration of specific primers from nucleic acids in a unique way andsuch primers can be subsequently assayed by polymerase extension todetermine the nature of the sequence at the 3′ termini of said primers.

1. A method for characterising nucleic acid molecules, which comprisesthe steps of: i) introducing a modified base which is a substrate for aDNA glycosylase into a DNA molecule by enzymatic extension of themolecule on a template nucleic acid; ii) excising the modified base bymeans of said DNA glycosylase so as to generate an abasic site; iii)cleaving the DNA at the abasic site so as to generate and release anextendible upstream DNA fragment having a 3′ hydroxyl terminus, whereinthe sequence of the extendible fragment is determined by the sequence ofthe template nucleic acid; and iv) incubating the released extendibleupstream DNA fragment in the presence of an enzyme allowing forextension thereof and a selected template nucleic acid, which haspartial or full sequence complementarity to said upstream DNA fragmentand analysing resultant fragment(s) to detect the presence or absence ofa mutation.
 2. A method according to claim 1, wherein the upstreamfragment is generated by cleaving the DNA at the 5′side of the abasicsite, such that the 3′terminus of the upstream fragment bears a hydroxylgroup.
 3. A method according to claim 2, wherein the cleavage isachieved with a 5′AP endonuclease.
 4. A method according to claim 1,wherein the upstream fragment is generated by cleaving at the 5′ side ofthe abasic site so as to leave a phosphate group at the 3′terminus ofthe upstream fragment and removing the phosphate group so that theupstream fragment bears a hydroxyl group at the 3′terminus.
 5. A methodaccording to claim 1, wherein the upstream fragment is generated bycleaving at the 3′side of the abasic site so as to generate adeoxyribose phosphate group at the 3′terminus of the upstream fragmentand subsequently removing the deoxyribose group to leave a hydroxylgroup at the 3′terminus.
 6. A method according to claim 1, wherein 5′deoxyribose moieties downstream of the 3′terminus of the upstreamfragment are removed so that the upstream fragment can be extended onthe template.
 7. A method according to claim 6, wherein the5′deoxyribose moieties are removed by a 5′deoxyribophosphodiesterase. 8.A method according to claim 1, wherein the modified base is introducedby enzymatic amplification of the DNA.
 9. A method according to claim 8,wherein the amplified strands are separated for a separate analysis ofthe respective strands.
 10. A method according to claim 8, wherein aprimer or one or more nucleotide (s) involved in the enzymaticamplification is labelled.
 11. A method according to claim 1, whereinthe enzyme is a polymerase.
 12. A method according to claim 11, whereinthe extendible upstream fragment is incubated in step iv) with thepolymerase in the presence of one or more nucleotide (s).
 13. A methodaccording to claim 12, wherein one or more of the nucloeotide (s) ofstep iv) is a dideoxy nucleotide.
 14. A method according to claim 12,wherein one or more of the nucleotide (s) of step iv) is labelled.
 15. Amethod according to claim 11, wherein the extension of step iv) isachieved by means of an amplification reaction using said extendible DNAfragment.
 16. A method according to claim 11, wherein the extension ofstep iv) is achieved by means of an amplification reaction including aprimer in addition to using said extendible DNA fragment.
 17. A methodaccording to claim 1, wherein the enzyme is a ligase.
 18. A methodaccording to claim 17, wherein the extendible upstream fragment isincubated with the ligase in the presence of a reporter oligonucleotide.19. A method according to claim 18, wherein the reporter oligonucleotideis partially degenerate.
 20. A method according to claim 1, wherein anyextended fragments resulting from step iv) are detected byhybridisation.
 21. A method according to claim 1, which is used todetect a known or unknown mutation.
 22. A method according to claim 1,wherein the method is used to analyse the CpG content of DNA bydetecting C to T transitions in DNA.